Prunus mume is a famous ornamental woody tree with colorful flowers. P. mume with yellow flowers is one of the most precious varieties. Regretfully, metabolites and regulatory mechanisms of yellow flowers in P. mume are still unclear. This hinders innovation of flower color breeding in P. mume. To elucidate the metabolic components and molecular mechanisms of yellow flowers, we analyzed transcriptome and metabolome between ‘HJH’ with yellow flowers and ‘ZLE’ with white flowers. Comparing the metabolome of the two varieties, we determined that carotenoids made contributions to the yellow flowers rather than flavonoids. Lutein was the key differential metabolite to cause yellow coloration of ‘HJH’. Transcriptome analysis revealed significant differences in the expression of carotenoid cleavage dioxygenase (CCD) between the two varieties. Specifically, the expression level of PmCCD4 was higher in ‘ZLE’ than that in ‘HJH’. Moreover, we identified six major transcription factors that probably regulated PmCCD4 to affect lutein accumulation. We speculated that carotenoid cleavage genes might be closely related to the yellow flower phenotype in P. mume. Further, the coding sequence of PmCCD4 has been cloned from the ‘HJH’ petals, and bioinformatics analysis revealed that PmCCD4 possessed conserved histidine residues, ensuring its enzymatic activity. PmCCD4 was closely related to PpCCD4, with a homology of 98.16%. Instantaneous transformation analysis in petal protoplasts of P. mume revealed PmCCD4 localization in the plastid. The overexpression of PmCCD4 significantly reduced the carotenoid content in tobacco plants, especially the lutein content, indicating that lutein might be the primary substrate for PmCCD4. We speculated that PmCCD4 might be involved in the cleavage of lutein in plastids, thereby affecting the formation of yellow flowers in P. mume. This work could establish a material and molecular basis of molecular breeding in P. mume for improving the flower color.

Prunus mume Siebold & Zucc (mei) is a horticulturally important fruit tree that undergoes anthesis in winter. Therefore, its flowering process is challenged by low-temperatures conditions. The transcription factor (TF) MYB21 is pivotal in regulating the flowering process, and particularly functions in petal expansion and filament elongation. However, the regulatory mechanism of PmMYB21 in mei remains unknown. To breed early-flowering cultivars, a deeper understanding of PmMYB21-regulated genes is essential. We employed DNA affinity purification sequencing (Dap-seq) to identify downstream genes bound by PmMYB21. The results revealed the promoter region is the primary binding region of PmMYB21, and the AGTTAGGTARR motif (motif1) is the predominant binding sequence type. Our analysis identified 8533 genes that are potentially bound by PmMYB21 with the motif1 sequence type, within the promoter region. These genes are involved in biological processes critical to flowering. Further refinement of candidate genes was achieved through Weighted Gene Co-expression Network Analysis (WGCNA), which identified the co-expressed genes of PmMYB21 during flowering activity. Integrating Dap-seq and WGCNA data, we narrowed down the candidate gene list to 54, with a focus on 4 MADS-box genes and 2 hormone signaling genes that are crucial to the flowering process under low-temperature conditions. This study offers valuable insights into the molecular underpinnings of PmMYB21’s role in the low-temperature flowering regulation of mei, paving the way for the development of new cultivars adapted to early blooming.